Noncoding SNPs decrease expression of FABP5 during COPD exacerbations

To the Editor: The pathogenesis of chronic obstructive pulmonary disease (COPD) is characterized by abnormal airway inflammation progressing to airflow obstruction and tissue destruction (1). While cigarette smoking and genetics clearly contribute to COPD risk, infections and associated exacerbations are also linked to disease pathogenesis (2). It is thought that systemic inflammation in COPD is a consequence of “spillover” of inflammatory mediators from the lungs. However, the mechanisms leading to the amplification of inflammation during episodes of exacerbation are largely unidentified. Fatty acid binding protein 5 (FABP5) is a small cytoplasmic protein involved in fatty acid transport and metabolism. We previously reported that FABP5 is downregulated in COPD and further downregulated in patients reporting one or more exacerbations (3). Here, we investigated the potential impact of genetic variation in FABP5. We performed a negative binomial analysis on the COPDGene SNP data set (https://www.copdgene.org/) and identified 5 linked single-nucleotide polymorphisms (SNPs) within the FABP5 locus that are significantly associated with severe exacerbations in a non-Hispanic White cohort (Figure 1A). To date, none of these SNPs (rs4338057, rs12549270, rs202275, rs202277, and rs202279) have been characterized to the best of our knowledge (Supplemental Table 1; supplemental material available online with this article; https://doi.org/10.1172/JCI175626DS1). The relative DNase sensitivity, enrichment for regulatory histone modifications, and overrepresentation of ENCODE-defined transcription factor binding sites in the rs4338057, […]

on ice.Chromatin was digested with micrococcal nuclease for 20 min at 37ºC with shaking at 850 rpm to yield 20% mononucleosomes.The reaction was stopped by the addition of 4 mM EGTA and incubated at 65°C for 10 min.Digested chromatin was washed twice in 50 mM NaCl, 10 mM Tris-HCl (pH 7.5), 10 mM MgCl2.The chromatin was then subjected to T4 Polynucleotide Kinase in 50 mM NaCl, 10 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 100 µg/mL BSA, 2 mM ATP and 5 mM DTT at 37ºC for 15 min prior to the addition of Klenow Fragment at 37ºC for 15 min to generate blunt ends.Labeling was performed by the addition of biotin-dATP, biotin-dCTP, dTTP and dGTP at 66 mM each and incubating at 25ºC for 45 min.Enzymes were inhibited by the addition of 30 mM EDTA and incubation at 65ºC for 20 min.Chromatin was washed once in 50 mM Tris-HCl (pH 7.5) and 10 mM MgCl2.DNA ends between crosslinked nucleosomes were ligated using T4 DNA ligase for 2.5 hours at room temperature.Biotin-dNTPs on the unligated ends were removed by exonuclease III at 37ºC for 15 min.The samples were reverse crosslinked by overnight incubation at 65ºC with 2 mg/mL Proteinase K and 1% SDS.DNA was purified using phenol:chloroform:isoamyl Alcohol twice, run on a 3% TBE NuSieve agarose gel and dinucleotide bands were extracted and purified using streptavidin beads.Standard library preparation protocol including end-repair, A-tailing and adapter ligation was performed using the NEBnext Ultra II kit.The sequencing library was amplified by Kapa HiFi PCR enzyme with the lowest possible number of cycles to reduce PCR duplicates.MicroC experiments were mapped to hg38 and downstream contact sites were identified using the package HiC-Pro (v2.11.4), which has dependencies of samtools (v1.1) and python (v2.7) and the python packages bx-python (v0.8.9), scipy (v1.2.3), pysam (v0.16.0.1), pandas (v0.24.2), and the iced normalization package (v0.5.7).The configuration for HiC-Pro specified to remove all singleton, multimapped, and duplicate reads.Iced-normalized contact counts output by HiC-Pro were visualized using the plotgardener library (v1.4.2) in R v4.2.3.

Donor samples
Blood samples from the COPDGene cohort were used in this study as previously described (4).COPDGene (www.COPDGene.org) is a National Heart Lung and Blood Institute-funded multicenter observational study designed to identify the genetic risk factors associated with COPD.Cases are diagnosed by post-bronchodilator spirometry as GOLD Stage II or greater (FEV1 < 80% predicted and FEV1/FVC < 0.7).COPD cases are between ages 45 and 80 and have at least a 10 pack-years of smoking history, and can be current or former smokers (5).The follow up study includes blood sample collection.Peripheral blood mononuclear cells (PBMCs) from 9 SNP carriers and 10 non-carriers were freshly isolated using CPT tubes (BD Biosciences, Vacutainer CPT tubes) and separated by density gradient centrifugation (Supplemental Table 2).After washing in HBSS, PBMCs were frozen and kept in liquid nitrogen until used to perform FABP5 mRNA quantification and metabolic assays.

Genotyping of donors
Genotyping quality control was performed following previously described guidelines (6, 7).10,503 DNA samples from COPDGene subjects, including duplicates and controls, were genotyped by Illumina (San Diego, CA) on the HumanOmniExpress array.GenomeStudio quality control, including manual review of cluster plots, was performed by Illumina.Genotype calls and intensities were exported for further quality control.Subjects were assessed for SNP missingness (>1.5%),SNP heterozygosity (6 standard deviations above the mean), chromosomal aberrations (analysis of B allele frequency), gender (X and Y chromosome intensity), and cryptic relatedness by estimated IBD (> 0.125).After genotype cleaning, an additional set of subjects were excluded based for ineligibility in the primary study, including the presence of other lung disease and inadequate smoking history.An additional ten subjects were dropped because of discordance in alpha-1 genotyping and plasma protein phenotyping results.
Principal component analysis was performed to identify racial mismatches and population outliers.
Autosomal SNPs present in the HapMap3 dataset with a minor allele frequency of > 5% and Hardy-Weinberg p-value > 0.01 were pruned in plink (8) using an initial r 2 of 0.12 across a 1500 SNP window, and further pruned using an r 2 of 0.05 within a 50 SNP window.Principal components were generated using EIGENSOFT 3.0 (9) and were assessed using both COPDGene and HapMap3 subjects.After removal of racial mismatches, 42 non-Hispanic whites were found to fall beyond six standard deviations on the first three principal components and were removed.No outliers were found in the African American subjects.A summary of the subject exclusions is shown in Supplemental Table 3.

SNP determination
Analyses were conducted using R. Severe exacerbations during longitudinal follow-up, defined as a COPD-related hospitalization or emergency department visit, were modeled with a negative binomial regression with offset for exposure time and a zero-inflation model to account for the excess number of subjects who reported no acute episodes of respiratory disease.Associations of exacerbations to SNPs were first determined without covariates.This model was applied to the subjects in the COPDGene study population with the clinical covariates of age, sex, percentpredicted FEV1, self-report of gastroesophageal reflux, St George's Respiratory Questionnaire (SGRQ), smoking status, and history of prior exacerbation.The zero-inflation component of the model included the clinical covariate percent-predicted FEV1 only.Clinical covariates were selected after reviewing the current literature on exacerbations; only clinical covariates that were significant in several cohort studies were used.
For each SNP, the model fitting returns an estimate, standard error, and p-value for the association of the SNP within the count model (negative binomial) and an estimate for the association of the SNP for zero counts within the zero-inflated component of the model (gamma parameter).The p-values were adjusted for multiple testing using the Benjamini-Hochberg method and false discovery rate (FDR) values > 0.05 were considered significant (Supplemental Table 1).

Microarray Analysis
We accessed Gene Array data available in the NCBI Gene Expression Omnibus (GEO Accession #GSE42057) from a study that measured the expression of 54,675 transcripts using Affymetrix Human Genome U133 plus 2.0 Gene Array (Affymetrix, Santa Clara, CA) (10).We specifically looked for FABP5 expression and segregated the data between SNP carriers (SNP+) and noncarriers (SNP-).

Metabolic Phenotyping
Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using the Agilent Seahorse XFe96 Bioanalyzer.PBMCs (20x10 4 per well) were plated in quadruplets onto Seahorse 96-well plates and pre-incubated in the indicated Seahorse XF RPMI media at 37ºC for 1 hr in the absence of CO2.OCR was measured under basal conditions and after sequential addition of Oligomycin, Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and Rotenone/Antimycin A. ECAR was measured under basal conditions in glucosedeprived media and after sequential addition of Glucose, Oligomycin, and 2-deoxy-D-glucose (2-DG) following manufacturer's instructions.Each measured value was reported on Wave software (Agilent Technologies) and normalized to the number of cells in each well.The cell count per well was determined by fluorescent cell counting using BioTek Cytation 1/5 instrument.

Statistical analysis
Data are expressed as mean ± SEM.Statistical tests were performed using Prism 8 software (GraphPad).Student's t test was used to compare two groups.Differences were considered statistically significant when p < 0.05.

Study approval
Human studies were approved by National Jewish Institutional Review Board.Written informed consent was received prior to participation.

Footnote: 0
absence of mutation, 1 presence of mutation on one allele, 2 presence of mutation on both alleles.

Table 2 .
Donor characteristics and SNP status.

Table 3 .
Summary of subject exclusions

Table 4 .
Summary of SNP marker exclusions